Synthetic organic insecticides are primarily nerve poisons acting on the cholinergic system (organophosphorus compounds and methylcarbamates), the voltage-gated sodium channel (pyrethroids and DDT), and the GABA-gated chloride channel (cyclodienes and other polychlorocycloalkanes). Potassium channels comprise a large and diverse group of integral membrane proteins that determine the level of excitability and repolarization properties of neurons and muscle fibers [B. Hille, Ionic Channels of Excitable Membranes, Sinauer, Sunderland, Mass. (1984)]. The multiple essential functions encoded by the potassium channels make them excellent targets for new pesticides and animal and human therapeutics. Potassium channel diversity in the fruitfly Drosophila melanogaster results from an extended gene family coding for homologous proteins. Six genes encoding potassium channels have been cloned from Drosophila melanogaster which account for a large part of the diversity of potassium currents observed in insect nervous tissue [A. Wei, M. Covarrubias, A. Butler, K. Baker, M. Pak, L. Salkoff, Science 248, 599-603 (1990), N. S. Atkinson, G. A. Robertson, B. Ganetzky, Science 253,551-555, (1991), J. Warmke, R. Drysdale, B. Ganetzky, Science 252, 1560-1564 (1991), A. Bruggemann, L. A. Pardo, W. Stuhmer, O. Pongs, Nature 365, 445-448 (1993)]. Shaker and Sha1 encode voltage-gated potassium channels with rapid current activation and inactivating properties. Shab and Shaw encode delayed rectifier channels, with slow inactivating (Shab) and non-inactivating (Shaw) properties. S1o encodes a calcium-activated potassium channel and eag encodes a voltage-gated channel permeable to both potassium and calcium which is modulated by cyclic AMP.
Modulation of cardiac action potential by compounds that effect the behavior of potassium channels may be a useful treatment for serious heart conditions. In this regard, each of the potassium channels cloned from insects have corresponding versions in mammalian species, including, specifically, a delayed rectifier potassium channel homolog, RAK, cloned from rat cardiac tissue [M. Paulmichl, P. Nasmith, R. Hellmiss, K. Reed, W. A. Boyle, J. M. Nerbonne, E. G. Peralta, D. E. Clapham, Proc. Natl. Acad. Sci USA 88, 7892-7895 (1991)]. Thus, the RAK channel represents an important target of new drugs for the control of heart failure. The delayed rectifier potassium current in heart cells regulates the duration of the plateau of the cardiac action potential by countering the depolarizing, inward calcium current. Delayed rectifier potassium currents characteristically are activated upon depolarization from rest, display a sigmoidal or delayed onset, and have a nonlinear, or rectifying, current-voltage relation. Several types of delayed potassium conductances have been identified in cardiac cells based on measured single-channel conductances. Heart rate and contractility are regulated by second messenger modification of delayed rectifier potassium conductances, and species differences in the shape of the plateau may be influenced by the type and level of channel expression.
On the basis of predicted membrane spanning topology, potassium channels may be subdivided into two distinct classes: voltage-gated, calcium-activated, and cyclic nucleotide-gated potassium channels that are composed of six membrane spanning domains (S1-S6) and a single pore forming domain (H5), and inward rectifying potassium channels that pass through the membrane twice and also contain a single pore forming region [Y. Kubo, E. Reuveny, P. A. Slesinger, Y. N. Jan, L. Y. Jan Nature 364, 802-806 (1993); Y. Kubo, T. J. Baldwin, Y. N. Jan, L. Y. Jan Nature 362, 127-133 (1993)]. Here, we report the cloning and functional expression in yeast of a novel Drosophila melanogaster potassium channel. Further, we identify a Caenorhabditis elegans homolog that constitutes the second member of a new family of potassium channels exhibiting a topological configuration unique among the known classes of potassium channels.
The yeast Saccharomyces cerevisiae is utilized as a model eukaryotic organism for the purpose of studying potassium transport mechanisms. Due to the ease with which one can manipulate the genetic constitution of the yeast Saccharomyces cerevisiae, researchers have developed a detailed understanding of many complex biological pathways, including potassium transport. In yeast, high affinity potassium uptake is performed by the product of the TRK1 gene [R. F. Gaber, C. A. Styles, G. R. Fink Mol. Cell. Biol. 8, 2848-2859 (1988)]. Mutant yeast strains lacking trk1 function are incapable of growing in medium lacking high concentrations of potassium. Since potassium transport mechanisms are present in organisms as divergent as yeast and man, one could predict that expression of heterologous potassium channels in mutant cells might replace trk1 function, and support growth on medium containing low potassium concentration. In this regard, plant potassium channels were shown to function in yeast and represent important targets for new herbicides [J. A. Anderson, S. S. Huprikar, L. V. Kochian, W. J. Lucas, R. F. Gaber, Proc. Natl. Acad. Sci USA 89, 3736-3740 (1992); H. Sentenac, N. Bonnaud, M. Minet, F. Lacroute, J.-M. Salmon, F. Gaynard, C. Grignon, Science 256, 663-665 (1992); D. P. Schachtman and J. I. Schroeder, Nature 370, 655-658]. Thus, we have employed this yeast expression system for cloning and expression of potassium channels from heterologous species, making it useful for discovery of new pesticides, and animal and human therapeutics. Discovery of such compounds will necessarily require screening assays of high specificity and throughput. For example, new pesticides directed at potassium channels require high selectivity for insect channels and low activity against non-insect species. Screening assays utilizing yeast strains genetically modified to accommodate functional expression of heterologous potassium channels offer significant advantages in this area.